Methods and Compositions for In Vivo Induction of Pancreatic Beta Cell Formation

ABSTRACT

Embodiments of the invention stimulate three levels of beta cell physiology: (i) glucose metabolism, (ii) membrane receptor function, and (iii) transcriptional factors that result in the in vivo formation of beta cells in the pancreas for the purpose of treating diabetes. In certain aspects, the methods include the integration of three levels of cellular physiology: metabolism, membrane receptor function, and gene transcription. The integration of multiple levels of cellular physiology produces a synergistic effect on beta cell formation.

PRIORITY CLAIM

This application is a non-provisional application claiming priority toU.S. provisional application 61/678,077 filed Jul. 31, 2012, which isincorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

A sequence listing required by 37 CFR 1.821-1.825 is being submittedelectronically with this application. The sequence listing isincorporated herein by reference.

BACKGROUND

Most medical drug treatments have utilized a reductionist approach: onemolecule for one cellular pathophysiological condition. Although thereductionist approach has proven successful for monogenic diseases, ithas failed for complex diseases. Physicians have recognized that acombination of approaches is required to treat complex disorders such astype 1 or type 2 diabetes. One treatment for diabetes is theadministration of insulin injections, which dates back to 1922. However,insulin injections do not stop the development of diabetic complications(e.g., retinopathy, neuropathy, nephropathy, cardiovascular disease, andstroke) in many type 1 and type 2 diabetic patients. The treatment costof these diabetic complications is enormous and contributes in a majorway to the increased cost health care in diabetic patients.

Although advances have been made in biomedical research, scientists andclinicians are still looking for effective treatments for diabetes. Incertain forms of diabetes beta cells are damaged, deficient, ordepleted. Potential treatments for diabetes include drug-based therapiesand cell-based therapies, both of which have their limitations.Drug-based therapies usually treat symptoms only and patients arechronically dependent on them. Cell-based therapies are hampered by thescarcity of cells and their source, immune rejection, and highmanufacturing and distribution costs.

Cell-based therapy is one approach to the treatment of diabetes andother conditions in which a reduction in pancreatic beta cell number orbeta cell function is causative or contributory (D'Amour et al., NatureBiotech 24:1392-1401, 2006; Kroon et al., Nature Biotech 26:443-452,2008). Multicomponent cocktails is one method for reproducing embryonicprecursors of beta cells, for example a cocktail of transcriptionalfactors has been used in stem cell research (Eminli et al., NatureGenetics 41:968-976, 2009) or a viral vector cocktail has been used morerecently in the mouse (Zhou et al., Nature 455: 627-632, 2008). Ingeneral these cells are not fully developed in their response toglucose, and although the cells contain and express insulin, they failto secrete insulin in the presence of glucose or in response to changesin glucose concentration.

Thus, there remains a need for methods of treating diabetes, such asproducing beta cells that express and secret insulin in vivo in asubject.

SUMMARY

The methods described herein induce pancreatic beta cell formation invitro or in vivo. In certain aspects the methods induce pancreatic betacell formation in adult subjects without dedifferentiating cells torecapitulate the embryonic pathway. In further aspects the methodsinduce pancreatic beta cell formation in cells that are at variousstages of differentiation. In other aspects the methods can be used toin vitro to induce beta cell formation. Certain embodiments of theapproach described herein specifically target the post-embryonicinduction of pancreatic beta cell formation without reproducing theembryonic formation process of the pancreas—the embryonic formationprocess leads to the generation of multiple pancreatic endocrine celltypes. The ability to generate new beta cells in vivo in adult subjectscan provide a novel therapeutic approach for the treatment of patientswith type 1 and 2 diabetes mellitus, as well as other types of diabetes.The ability to increase the number of pancreatic beta cells in adultsubjects can be therapeutic, prophylactic, and/or curative in regards todiabetes.

Certain embodiments are directed to compositions and methods thatmodulate and integrate three levels of beta cell physiology: (i) glucosemetabolism, (ii) membrane receptor function, and (iii) transcriptionalfactors. In certain aspects, the methods described herein targetpost-embryonic induction processes of pancreatic beta cell formation.Since the embryonic process leads to multiple endocrine cell types, thepost-embryonic methods described herein are designed to induce primarilyor only the formation of beta cells. In certain aspects beta cells areformed in vivo in organs or tissues, such as the pancreas, or in vitrowithout causing formation of detectable levels of other endocrine celltypes (e.g., alpha cells that secrete glucagon or delta cells thatsecrete somatostatin). The inventors are not aware of any reports inwhich pancreatic beta cell formation is induced in vivo in an adultsubject without inducing other pancreatic endocrine cells types. Thisability to generate beta cells in vivo in adult subjects provides anovel therapeutic approach for the treatment of patients with type 1 and2 diabetes mellitus, as well as other types of diabetes.

Certain embodiments employ a gene transfer approach to modulateintracellular targets for pancreatic beta cell formation. Otherembodiments use therapeutic agents that mimic the cellular processmodulated by the gene transfer methodology. Still other embodiments usea combination of gene transfer and therapeutic agents.

In certain aspects, glucokinase (GK) (GenBank Accession No.NP_(—)034422.2 (GI:31982798) or NP_(—)000153.1 (GI:4503951), which isincorporated herein by reference in its entirety as of the applicationfiling date), functional segments or variants thereof, or an activatorof GK activity is provided to increase the glucose metabolic rate. Useof other GK nucleic acids transcribed from the GK gene (see GenBankaccession NG_(—)008847.1, which is incorporated herein by reference) isalso contemplated. In certain aspects a variant of GK that maintains GKenzymatic activity can also be used. In a further aspect, an inhibitorof protein tyrosine phosphatase 1B (PTB1B) (e.g., an inhibitory RNA,anti-sense DNA, small molecule inhibitor, etc.) is provided to increasetyrosine kinase receptor or tyrosine kinase associated receptoractivity. In still a further aspect, Pdx-1 (GenBank Accession No.NP_(—)000200 (GI:4557673), which is incorporated herein by references asof the application filing date), a functional segment or variantthereof, or an activator of Pdx-1 activity is provided to target genesinvolved in beta cell formation. In certain aspects a variant of Pdx-1that maintains Pdx-1 transcription activating abilities can also beused. Use of other Pdx-1 nucleic acids transcribed from the Pdx-1 gene(see GenBank accession NG_(—)008183) is also contemplated. In certainaspects, a nucleic acid encoding a protein of interest is administered.In a further aspect, each protein or inhibitor is comprised in anindividual and separate expression cassette or expression vector. Inother aspects, two or more proteins are encoded in a single expressioncassette or expression vector.

In certain aspects the beta cell inducing agent(s) are administereddirectly to the pancreas. In certain aspects, the beta cell inducingcomposition(s) are administered via the pancreatic duct. In a furtheraspect, beta cell inducing agents are administered orally orintravascularly.

In a further aspect the beta cell inducing agent(s) are administered toa cell in vitro. In certain aspect the cell treated in vitro are cellsthat are heterologous or autologous to the subject being treated. In oneaspect autologous cells are isolated from a patient, administered theinducing agent(s), and the in vitro treated cells are then implanted inthe patient. In other aspects a heterologous cell is obtained,administered the inducing agent(s), and the in vitro treated cells arethen implanted in the patient.

In certain aspects, an organ, tissue, or cell target is one that can beinduced to sense glucose level and secrete insulin. In certain aspects,a target cell or tissue exhibits the ability to induce or be engineeredfor expression of Glut 2 and/or Glucokinase; expression of proinsulin;and expression of protein convertases to cleave the proinsulin. Cellsare present in the human body that have at least two characteristics ofa beta cell. A gut K cell is one example of such a cell. Gut K cellsexpress Glut 2, glucokinase, and protein convertase, thereforeinducement of insulin expression is needed. In another example, livercells also express Glut 2 and glucokinase.

Certain embodiments are directed to methods of inducing beta cellformation from post-embryonic pancreatic cells in vivo. In certainaspects, the method includes providing to a pancreas in vivo, acombination of (i) a first agent that increases glucokinase (GK) levelsor activity, (ii) a second agent that increases tyrosine receptor kinaseactivity, and (iii) a third agent that increases Pdx-1 mediatedtranscription.

In certain aspects the first agent is a nucleic acid encodingglucokinase. The nucleic acid encoding glucokinase can be incorporatedin a viral vector. In certain aspects, the viral vector is a lentivirusvector or other nucleic acid delivery vector or particle. The nucleicacid can comprise a posttranscriptional regulatory element 3′ of thecoding sequence, e.g., a posttranscriptional regulatory element ofwoodchuck hepatitis virus (WPRE). In certain embodiments a polypeptidecomprising a protein transduction domain can be administered to a cellor subject. In certain aspect glucokinase is provided as a recombinantprotein fusion comprising protein transduction domains. Proteintransduction domains (PTDs or cell permeable proteins (CPP) or membranetranslocating sequences (MTS)) are small peptides that are able to ferrymuch larger molecules into cells independent of classical endocytosis.Many known PTDs bind to the same surface molecules (Heparan SulphateProteoglycans, HSPG) before internalization, and that internalization isdependent on these molecules. In further aspects, the first agent can bea small molecule activator of glucokinase. An activator of glucokinasecan include, but is not limited to R1440, RO0281675, RO4389620(Piragliatin), LY2121260, PSN-GK1, or GKA-50.

In certain aspects, the second agent is an inhibitor of protein tyrosinephosphatase 1B. The protein tyrosine phosphatase 1B inhibitor can be anshRNA inhibitor of protein tyrosine kinase phosphatase 1B. In certainaspects, the protein tyrosine phosphatase 1B inhibitor can be, but isnot limited to Wyeth Research Inc., 32D; antisense ISIS-PTP1BRX; AbbottLaboratories, Inc., Isoxazole™; Abbott Laboratories, Inc., antisenseoligonucleotides designed to downregulate expression of PTP1B; MerckFrosst Center for Therapeutic Research, selective inhibitors of PTP1Bcompound 1 and 3; Incyte Corporation, Inc., (S)-isothiazolidinone((S)-IZD) heterocyclic phosphotyrosine; or Affymax, Inc., triarylsulfonamide based PTP1B inhibitors.

In still further aspects, the third agent is a beta cell selectivetranscriptional activator. In certain aspects the transcriptionalactivator is a nucleic acid encoding Pdx-1. In certain aspect atranscriptional activator is administered to a cell or subject as arecombinant protein fusion with a protein transduction domain. Incertain aspects other transcriptional activators used alone or incombination with one or more of NeuroD, Isl1, Nkx6.1, and/or Pax4 can beused. In a further embodiment, the compound troglitazone can be providedin place of or in conjunction with Pdx-1 transcriptional activation.

In certain aspects, the first, second, and third agents are provided ina single composition. In another aspect, the first, second, and thirdagents are provided separately. The agents can be administered almostsimultaneously or within a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minute(s) orhour(s) administration window. In certain embodiments the first, second,and third agent are provided sequentially. In other embodiments thefirst, second, and third agent are provided simultaneously. In certainembodiments, the first and second agents, first and third agents, or thesecond and third agents are the same agent.

In certain aspects, the first, second, and third agent are provided byinjection or infusion into the pancreas, or other target organ ortissue. In a further aspect, injection or infusion into the pancreas isthrough the pancreatic duct.

Other embodiments include methods of treating diabetes comprising:providing a therapeutic composition to a pancreas or other organ ortissue in vivo comprising the agents described above. In certain aspectsthe therapeutic composition comprises (i) glucokinase expressioncassette configured to express a functional glucokinase protein, (ii) atyrosine phosphatase 1B inhibitor, and (iii) a Pdx-1 expression cassetteconfigured to express a functional Pdx-1 protein, wherein pancreaticbeta cells are induced. In a further embodiment, the compoundstroglitazone can be provided in conjunction with Pdx-1.

Certain embodiments include methods of treating diabetes comprising:obtaining a target cell heterologous to a patient or isolating aautologous target cell from a patient and providing a therapeuticcomposition to the cell in vitro comprising the agents described above.In certain aspects the therapeutic composition comprises (i) glucokinaseexpression cassette configured to express a functional glucokinaseprotein, (ii) a tyrosine phosphatase 1B inhibitor, and (iii) a Pdx-1expression cassette configured to express a functional Pdx-1 protein,wherein pancreatic beta cells are induced. In a further embodiment, thecompounds troglitazone can be provided in conjunction with Pdx-1. Themethods further comprise implanting the treated target cell in apatient.

In certain aspects, one, two, or more nucleic acids (i.e., genes) can beused. In certain aspects, three nucleic acids are used. In a furtheraspect, one, two, or three nucleic acids can be combined with one ormore chemical agent. In still further aspects, chemical agents thatpositively or negatively modulate the target pathways can be usedwithout nucleic acids.

In certain embodiments, chemical agent combinations can include, but arenot limited to chemical agent activators of GK in combination withchemical agent inhibitors of PTB1B and/or chemical agent activators ofPdx-1; or chemical agent activators of Pdx-1 with chemical agentinhibitors of PTB1B.

In certain embodiments, nucleic acids can be used in combination withchemical agents. In certain aspects, one or more of a GK gene, PTB1Binhibitory nucleic acid, and/or a Pdx-1 activating nucleic acid can beused in combination with one or more chemical agent PTB1B inhibitor,chemical agent GK activator, and/or chemical agent Pdx-1 activator. Asused herein, the gene can refer to a nucleic acid encoding a therapeuticnucleic acid such as GK gene encoding the GK enzyme, the PTBIB geneencoding an inhibitory nucleic acid, or a Pdx-1 encoding an activator ofthe Pdx-1 pathway.

Various combinations of agents include, but are not limited to chemicalagent GK activator(s)+chemical agent PTP1B inhibitor(s); chemical agentGK activator(s)+chemical agent PTP1B inhibitor(s)+chemical agent Pdx-1activator(s); chemical agent Pdx-1 activator(s)+chemical agent PTP1Binhibitor(s); GK gene+PTP1B gene; GK gene+chemical agent PTP1Binhibitor(s); GK gene+PTP1B gene+chemical agent PTP1B inhibitor(s); GKgene+chemical agent GK activator(s)+PTP1B gene; GK gene+chemical agentGK activator(s)+PTP1B gene+chemical agent PTP1B inhibitor(s); GKgene+chemical agent GK activator(s)+chemical agent PTP1B inhibitor(s);chemical agent GK activator(s)+PTP1B gene; chemical agent GKactivator(s)+chemical agent PTP1B inhibitor(s); chemical agent GKactivator(s)+PTP1B gene+chemical agent PTP1B inhibitor(s); GK gene+PTP1Bgene+Pdx-1 gene; GK gene+chemical agent GK activator(s)+PTP1B gene+Pdx-1gene; GK gene+chemical agent GK activator (s)+PTP1B gene+chemical agentPTP1B inhibitor(s)+Pdx-1 gene; GK gene+chemical agent GKactivator(s)+PTP1B gene+chemical agent PTP1B inhibitor(s)+Pdx-1gene+chemical agent Pdx-1 activator; GK gene+chemical agent GKactivator(s)+PTP1B gene+chemical agent PTP1B inhibitor(s)+chemical agentPdx-1 activator; chemical agent GK activator(s)+PTP1B gene+Pdx-1 gene;chemical agent GK activator(s)+PTP1B gene+chemical agent PTP1Binhibitor(s)+Pdx-1 gene; chemical agent GK activator(s)+PTP1Bgene+chemical agent PTP1B inhibitor(s)+Pdx-1 gene+chemical agent Pdx-1activator(s); chemical agent GK activator (s)+chemical agent PTP1Binhibitor(s)+chemical agent Pdx-1 activator(s); chemical agent GKactivator (s)+chemical agent PTP1B inhibitor(s)+chemical agent Pdx-1activator(s)+PTP1B gene; GK gene+chemical agent GK activator(s)+chemicalagent PTP1B inhibitor(s)+chemical agent Pdx-1 activator (s); GKgene+chemical agent GK activator(s)+chemical agent PTP1Binhibitor(s)+PTP1B gene; GK gene+chemical agent GK activator(s)+chemicalagent PTP1B inhibitor(s)+Pdx-1 gene; Pdx-1 gene+PTP1B gene; Pdx-1gene+chemical agent PTP1B inhibitor(s); chemical agent Pdx-1activator(s)+PTP1B gene; chemical agent GK activator(s)+Pdx-1gene+chemical agent PTP1B inhibitor(s); chemical agent Pdx-1activator(s)+Pdx-1 gene+PTP1B gene; chemical agent Pdx-1activator(s)+Pdx-1 gene+chemical agent PTP1B inhibitor(s); PTP1Bgene+chemical agent GK activator(s)+chemical agent Pdx-1 activator(s);chemical agent PTP1B inhibitor(s)+Pdx-1 gene+GK gene; chemical agentPTP1B inhibitor(s)+Pdx-1 gene+PTP1B gene; chemical agent PTP1Binhibitor(s)+chemical agent Pdx-1 activator(s)+GK gene; chemical agentPTP1B inhibitor(s)+chemical agent Pdx-1 activator(s)+PTP1B gene;chemical agent PTP1B inhibitor(s)+chemical agent GK activator(s)+PTP1Bgene; GK gene+chemical agent GK activator(s)+Pdx-1 gene+chemical agentPTP1B inhibitor(s); chemical agent GK activator(s)+Pdx-1 gene+chemicalagent PTP1B inhibitor(s)+PTP1B gene; GK gene+Pdx-1 gene+chemical agentPdx-1 activator(s)+PTP1B gene; GK gene+Pdx-1 gene+chemical agent Pdx-1activator(s)+chemical agent PTP1B inhibitor(s); GK gene+chemical agentPdx-1 activator(s)+PTP1B gene+chemical agent PTP1B inhibitor(s); GKgene+chemical agent GK activator(s)+Pdx-1 gene+chemical agent Pdx-1activator(s)+PTP1B gene; GK gene+chemical agent GK activator(s)+Pdx-1gene+chemical agent Pdx-1 activator(s)+chemical agent PTP1Binhibitor(s); GK gene+Pdx-1 gene+chemical agent Pdx-1 activator(s)+PTP1Bgene+chemical agent PTP1B inhibitor(s); Pdx-1 gene+chemical agent Pdx-1activator(s)+PTP1B gene+chemical agent PTP1B inhibitor(s); or Pdx-1gene+PTP1B gene+chemical agent PTP1B inhibitor(s)+GK gene.

In certain embodiments, a single agent can (i) positively modulateglucokinase activity, and positively modulate tyrosine kinase receptoractivity and/or tyrosine kinase associated receptor activity; (ii)positively modulate glucokinase activity, and positively modulate betacell specific transcription; or (iii) positively modulate tyrosinekinase receptor activity and/or tyrosine kinase associated receptoractivity (e.g., inhibit PTB1B), and positively modulate beta cellspecific transcription.

In certain aspect chemical agent GK activator(s) can act at two levelsincreasing the glucose metabolism rate and increasing the Pdx-1 mediatedgene expression. Chemical agent PTB1B inhibitor(s) in combination withchemical agent GK activator(s) can target each of the three pathwaysdescribed herein.

In certain aspects, in disease states such as type II diabetes chemicalagent PTP1B inhibitor(s) can act on both the tyrosine kinase receptorlevel and GK levels in the presence of insulin. In certain aspects a GKactivator(s) can increase glucose metabolism and Pdx-1 mediatedtranscriptional activation. For example, Rosiglitazone increases theexpression of GK and Pdx-1 mediated effects. Chemical agent PTP1Binhibitor(s) in combination with insulin secretion competence canincrease glucose metabolism and increase tyrosine kinase receptoractivity. Furthermore, the family of PPAR-gamma activator(s) likeRosiglitazone increases GK expression and Pdx-1 expression. Thus, asingle agent can be administered to modulate multiple target pathways.

As used herein “target cell” and “target cells” refer to precursorcells, isolated cells, stem cells, cells of the pancreas or other organsor tissues that can be induced to form beta cells or beta cell-likecells. The cells can be beta cells or non-beta cells prior toinducement. A precursor cell is a cell that is not fully differentiated.

As used herein, expression refers to mRNA levels (nucleic acidexpression) and/or protein levels (protein expression). Oligonucleotidessuitable to detect mRNA, e.g., using RT-PCR, can be designed usingtechniques routine in the art. Alternatively or in addition, proteinexpression can be assessed using any art-recognized technique (e.g., anyantibody based detection technique).

As used herein, the term “treatment,” when used in the context of atherapeutic strategy to treat a disease or disorder means any manner inwhich one or more of the symptoms of a disease or disorder areameliorated or otherwise beneficially altered. As used herein,amelioration of the symptoms of a particular disease or disorder refersto any lessening, whether permanent or temporary, lasting or transientthat can be attributed to or associated with treatment by thecompositions and methods of the present invention.

The terms “effective amount” and “effective to treat,” as used herein,refer to an amount or a concentration of one or more compounds or apharmaceutical composition described herein utilized for a period oftime (including in vivo acute or chronic administration, and periodic orcontinuous administration) that is effective within the context of itsadministration for causing an intended effect or physiological outcome.

Effective amounts of one or more compounds, or a pharmaceuticalcomposition for use in the present invention include amounts thatpromote beta cell formation or maturity, e.g., an increase inglucose-dependent insulin secreting cells or an increase inglucose-dependent secretion from a cell.

The term “subject” is used throughout the specification to describe ananimal, human or non-human, to whom treatment according to the methodsof the present invention is provided. In certain aspects, the subject ishuman.

The term “providing” is used according to its ordinary meaning “tosupply or furnish for use.” In some embodiments, a protein is provideddirectly by administering the protein, while in other embodiments, theprotein is provided by administering a nucleic acid that encodes theprotein. In other embodiments, an inhibitor such as an shRNA can beprovided to reduce protein levels in a cell. In certain aspects theinvention contemplates compositions comprising various combinations oftherapeutic nucleic acids, peptides, and/or small molecules.

Other embodiments of the invention are discussed throughout thisapplication. Any embodiment discussed with respect to one aspect of theinvention applies to other aspects of the invention as well and viceversa. Each embodiment described herein is understood to be embodimentsof the invention that are applicable to all aspects of the invention. Itis contemplated that any embodiment discussed herein can be implementedwith respect to any method or composition of the invention, and viceversa. Furthermore, compositions and kits of the invention can be usedto achieve methods of the invention.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional elements or methodsteps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofthe specification presented herein.

FIG. 1. An example of an approach using an induction cocktail comprisingthree molecules to induce pancreatic beta cells formation in vivo in theadult pancreas.

FIG. 2A-2C. Illustrates the design and validation of Lentiviralconstructs, (A) glucokinase, (B) Pdx-1, and (C) shRNA PTP1B.

FIG. 3. In vivo over-expression of PDX-1 and GK, and suppression ofPTP1B expression by the CNIP cocktail (Lenti-GCK+Lenti-Pdx-1 andLenti-shRNA PTP1B).

FIG. 4. Quantitation of single beta cell staining in the adultpancreatic tissue in mice injected with a beta cell formation cocktailcompared with a control group injected with placebo cocktail.

FIG. 5. An example of a beta cell formation cocktail comprising threemolecules (GK, PTP1B inhibitor, and Pdx-1) induced proliferation in theadult mouse pancreas compared with the control adult mouse groupinjected with placebo.

FIG. 6. Pancreatic beta cell mass was significantly increased in adultmice injected with a beta cell formation cocktail (GK, PTP1B inhibitor,and Pdx-1) compared with the control adult mice group injected with theplacebo. Immunofluorescence images of insulin staining were capturedusing confocal microscopy. The beta cell and total pancreatic areas werequantified with Image J (NIH, Bethesda Md.). Total beta cell mass wascalculated as the total beta cell area expressed as a percentage of thetotal area of the pancreas.

FIG. 7. Illustrates the number of beta cell clusters in the pancreas(cluster density) in the adult mouse group injected with the beta cellformation cocktail compared with the control adult mouse group injectedwith the placebo. Cluster density was determined as the number of betacell clusters divided by the total area of the pancreas.

FIG. 8. Illustrates the fasting plasma insulin concentration in theadult mouse group injected with a beta cell formation cocktail (GK,PTP1B inhibitor, and Pdx-1) compared with the adult mouse control groupinjected with placebo.

FIG. 9 BrdU marker of proliferation in islets and exocrine tissue 4weeks post-injection of the cocktail (Lenti-GCK+Lenti-Pdx-1+Lenti-shRNAPTP1B) or by two molecules or each molecule individually. The figure isrepresentative of an area of the pancreas examined in ten sections peranimal (n=3 to 4 for each group), separated by 200 μm. The results areexpressed as the fold-increase in number of BrdU-labeled cells comparedwith controls. Confocal laser microscopy was used for analysis.Lenti=Lentivirus; GCK=glucokinase; PTP1B=protein-tyrosine phosphatase1B. Data are presented as mean±SE. *=p<0.001 CNIP cocktail vs control;=p<0.05 CNIP cocktail vs [GK+PTP1B]; #=p<0.01 [GK+PTP1B] vs control.

FIG. 10 Beta cell mass 4-weeks post-injection with the cocktail CNIP(Lenti-GCK+Lenti-Pdx-1+Lenti-shRNA PTP1B) or by two molecules or eachmolecule individually compared to cocktail control and each other (n=3to 4 for each group). Total pancreatic and insulin positive stainingareas of each section were measured using Image J (NIH, Bethesda, USA).Beta cell mass was calculated as the ratio of total insulin positivearea to total pancreatic area of all sections, multiplied by thepancreatic tissue wet weight. The figure is representative of an area ofthe pancreas examined in ten sections per animal, separated by 200 μm.Confocal laser microscopy was used for analysis. Lenti=Lentivirus;GCK=glucokinase; PTP1B=protein-tyrosine phosphatase 1B. *=p<0.001=CNIPcocktail vs control; p<0.001 CNIP cocktail vs [GK+Pdx-1]; p<0.001 CNIPcocktail vs [PTP1B+Pdx-1]; ▪=p<0.05 CNIP cocktail vs [GK+PTP1B].#=p<0.05 [GK+PTP1B] vs [PTP1B+Pdx-1); p<0.05 [GK+PTP1B] vs control. Dataare presented as mean±SE.

DESCRIPTION

Certain methods described herein represent a concept that contradictsthe scientific doctrine of one molecule to one cellular control process.In certain aspects, the methods include the integration of three levelsof cellular physiology: metabolism, membrane receptor function, and genetranscription. The integration of multiple levels of cellular physiologyproduces a synergistic effect on beta cell formation. Synergy requiresthat multiple molecules work together to produce an effect that isgreater than the sum of their individual effects. Using the synergisticapproach described herein, the inventors have successfully inducedpancreatic beta cell formation in the adult pancreas. The ability togenerate beta cells in vivo in adult animals and humans provides a noveltherapeutic approach for the treatment of subjects with type 1 and type2 diabetes mellitus.

I. METHODS OF TREATING DIABETES

The inventors have demonstrated that, utilizing “Cellular NetworkingIntegration & Processing” (CNIP), pancreatic beta cell formation can beincreased in vivo in adult subjects. According to the CNIP approach, theinventors intervene at three major levels in cell processing: (1) first,at the level of intracellular carbohydrate metabolism, (2) second, atthe level of the membrane receptor function, (3) third, at the level ofgene expression. By targeting all three levels, one can generate asynergistic interaction that induces beta cell formation. The inventorsrefer to one example of this method as “Syner-III,” with Syner being theprefix from the Greek name synergos and III is Roman number three.

The CNIP approach is designed to mimic the formation of beta cells inadult subjects and not to reprogram the cell at the stage of embryonicdevelopment. The inventors note that the cocktail of transcriptionalfactors used in stem cell research or in the viral vector cocktail usedmore recently in the mouse model (Zhou et al., Nature 455: 627-32, 2008)are used to generate beta cells by reproducing the embryonic stage ofdevelopment. In contrast, the CNIP approach is designed to act in theadult state and utilizes a mechanism that integrates the three levels ofcellular regulation to induce beta cell formation.

The methods described herein induce pancreatic beta cell formation invivo in adult subjects without dedifferentiating cells to recapitulatethe embryonic pathway. The CNIP approach specifically targets thepost-embryonic induction of pancreatic beta cell formation withoutreproducing the embryonic formation process of the pancreas—theembryonic formation process leads to the generation of multiplepancreatic endocrine cell types. The ability to generate new beta cellsin vivo in adult subjects can provide a novel therapeutic approach forthe treatment of patients with type 1 and 2 diabetes mellitus, as wellas other types of diabetes. The ability to increase the number ofpancreatic beta cells in adult subjects can be therapeutic,prophylactic, and/or curative in regards to diabetes.

In certain embodiments, compositions and methods described herein can beapplied to tissues other than the pancreas. In certain aspects,compositions described herein can be delivered into the gut endocrine Kcells and be an able to form insulin-like beta cell that would secreteinsulin in response to an elevation of blood glucose. In a furtheraspect, the compositions described herein can be delivered to the liverto induce formation of beta cells that respond to glucose. In stillother aspects, the compositions described herein can be applied at thelast step of stem cell differentiation and/or dedifferentiation to formbeta cells. In certain aspects, the compositions described herein can bedelivered to various cells and/or tissues in the body to form beta cellsand therefore, are not limited to the specific examples describedherein.

In certain embodiments target cells are treated in vitro. Target cellsare those cells that have the capability or can be induced to have thecapability of forming beta cells. Methods for providing or obtainingsuch target cells are known in the art and include either providingtissue containing target cells and isolating the target cells by methodsknown in the art, e.g. with the help of cell surface specific antibodiesand using a FACS (cell sorter) or cultivation of the cells underspecific conditions allowing the growth of target cells. In certainaspects there are suitable target cell lines (Lieber et al., Int JCancer 15(5):741-47, 1975).

Any cell being capable of differentiating into pancreatic beta cells canbe used as a target cell of the method of the invention. This includesprecursor cells derived from human or animal (e.g., mammal) tissue. Incertain embodiments the target cell is an autologous target cell, i.e.,it contains the same genetic information as cells of the subject beingtreated. In certain aspects the target cell has not been geneticallymodified prior to the treatment being administered. In certain aspects atarget cell is selected from the group consisting of a pancreaticprecursor cell, a small intestine precursor cell, a liver precursorcell, a precursor cell derived from the pancreatic duct population,precursor of neuroendocrine cell, and a pancreatic stem cell. Thisincludes all somatic differentiated cells from a human or animal tissue.In certain aspects a target cell is selected from the group consistingof somatic differentiated cell from the liver, endocrine gut cell,pancreatic duct cell, exocrine and endocrine pancreatic cell, andneuroendocrine cell

Once target cells have been obtained or provided, the cells can be grownand manipulated in an in vitro cell culture system, which includesstandard cell culture systems like tissue culture dishes and 6-well,24-well or 96-well plates. Culture conditions will depend on the targetcell and the person skilled in the art will know how to cultivate thecells.

A. Glucose Metabolism

Glucose metabolism is the first aspect in the CNIP approach to inducingbeta cell formation in the adult pancreas or other organs or tissues.Glucose is the major energy source utilized by the mammalian cell, andmetabolism of glucose provides the energy for cellular function andproliferation (Bohnsack and Hirschi, Annu Rev Nutr. 24: 433-453, 2004).Inhibition of glycolysis stops cell cycle progression, documenting thenecessity of glucose metabolism to induce proliferation (Newcomb et al.,Eukaryot. Cell. 2:143-149, 2003). Factors that induce pancreatic betacell formation in vivo include an increase in glucose metabolism(Bernard et al., FASEB J. 13:1195-1205, 1999; Alonso et al., Diabetes56:1792-1801, 2007). Glucose infusion in adult rats for a period of only24 h increased beta cell number by ˜50% (Bernard et al., FASEB J.13:1195-1205, 1999). Furthermore, glucose promotes beta cell survival bysuppressing a constitutive apoptotic program in vitro (Hoorens et al.,J. Clin. Invest. 98:1568-1574, 1996). Glucose metabolism primes thepancreas for induction of pancreatic beta cell formation.

In certain aspects, the rate of glucose metabolism is increased byproviding a nucleic acid encoding glucokinase, or increasing theactivity of glucokinase or other enzymes or regulators. In certainembodiments the functions ascribed to the nucleic acid described hereincan be provide by administering various chemical compounds or smallmolecules that increase glucose metabolism. Glucokinase activatingcompounds include, but are not limited to Roche Inc., compound R1440;Hoffman-La Roche Inc., compound RO0281675; Hoffman-La Roche Inc.,compound RO4389620 (Piragliatin); Eli Lilly Inc., compound LY2121260;OSI Pharmaceuticals, Inc., compound PSN-GK1; Astra-Zeneca, Inc.,compound GKA-50; Pfizer Inc., glucokinase activators described inInternational Patent publication WO/2007122482); Merck-Banyu Inc.,glucokinase activators described in International Patent publicationWO/2003080585; Takeda Inc., glucokinase activators described inInternational Patent publication WO/200710434); Johnson & Johnson Inc.,glucokinase activator described in International Patent publicationWO/2007075847); and the like.

B. Receptor Tyrosine Kinases and Tyrosine-Kinase-Associated receptors.

Membrane receptor tyrosine kinase(s) and/or tyrosine-kinase-associatedreceptors are a second component of the CNIP approach to induce theformation of pancreatic beta cells in an adult subject. The secondaspect in the generation of pancreatic beta cells following aphysiological stimulus is for the cell to receive the message throughits membrane receptors. The membrane receptors responsible for thestimulation of pancreatic beta cell mass are from the tyrosine kinasefamily of receptors and tyrosine-kinase-associated family of receptors.During pregnancy the pancreatic beta cell mass increases in response tothe development of insulin resistance and increased fetal/placentaenergy demand and this effect is mediated by increased prolactin,estrogen, and placental lactogen secretion (Heit et al., Annu. Rev CellDev. Biol. 22:311-338, 2006). The failure of the beta cell to compensateby augmenting its secretion of insulin leads to gestational diabetes.Islet enlargement and beta cell hyperplasia have been observed inautopsied pregnant humans (Van Assche et al., Br. J. Obstet Gynaecol 85:818-820, 1978). The hormonal stimuli (prolactin, estrogen, and placentallactogen) during pregnancy to increase the pancreatic beta cell mass actthrough tyrosine kinase associated receptors (Nielsen et al., Diabetes50(Suppl. 1): S25-S29, 2001). Other hormones that increase beta cellmass also act through the tyrosine kinase family of receptors andinclude hepatocyte growth factor, platelet-derived growth factor, growthhormone, insulin, IGF-1 and EGF (Nielsen et al., Diabetes 50(Suppl. 1):S25-S29, 2001). Of note, the effect of these hormones on beta cell massalso relies on glucose metabolism. In the absence of glucose, theability of hormones acting through the tyrosine kinase family toincrease pancreatic beta cells mass is lost (Cousin et al., Biochem. J.344:649-658, 1999). Consequently, the CNIP approach combines the effectof glucose metabolism, and membrane tyrosine kinase(s) and/ortyrosine-kinase(s) associated receptors to induce beta cell formation inthe adult pancreas.

In certain embodiments the function(s) ascribed to the nucleic acidsdescribed herein can be provided by administering various chemicalcompounds or small molecules that increase tyrosine kinase receptorand/or tyrosine-kinase(s) associated receptor activity for beta cellformation in an adult pancreas. In certain aspects, inhibitory nucleicacids such as anti-sense DNA or inhibitory RNA molecules can be used.Such compounds include, but are not limited to PTP1B inhibitor compoundssuch as Wyeth Research Inc., 32D; antisense ISIS-PTP1BRX; AbbottLaboratories, Inc., Isoxazole; Abbott Laboratories, Inc., antisenseoligonucleotides designed to downregulate expression of PTP1B; MerckFrosst Center for Therapeutic Research, selective inhibitors of PTP1Bcompound 1 and 3; Incyte Corporation, Inc., (S)-isothiazolidinone((S)-IZD) heterocyclic phosphotyrosine; Affymax, Inc., triarylsulfonamide based PTP1B inhibitors; and the like.

Other shRNA targeting tyrosine phosphatase family proteins can beincluded alone or in combination with shRNA PTP1B. PTP1B acts on themajority of the tyrosine kinase family of receptors that have beenimplicated in pancreatic beta cell function in the adult pancreas.However, T-cell protein tyrosine phosphatase (TCPTP) and SHP-2, twoother members of intracellular protein phosphatase, have been shown totarget receptor tyrosine kinases implicated insulin signaling (Tonks,Nat Rev. Mol Cell Biol 7:833-46, 2006). SHP-2 (SH2-domain containingphosphatase-2) is a ubiquitously expressed intracellular proteintyrosine phosphatase that contains two amino-terminal Src homology 2(SH2) domains. SH2-2 binds to both the tyrosine-phosphorylated insulinreceptor and IRS-1. TCPTP exists in two forms: an endoplasmicreticulum-targeted 48-kDa from (TC48) and a nuclear 45-kDa form (TC45).TC-PTP has been demonstrated to negatively regulate insulin signalingand the prolactin receptor (Aoki and Matsuda, J. Biol. Chem.275:39718-26, 2000; Tonks, Nat Rev. Mol Cell Biol 7:833-46, 2006).Therefore, nucleic acids expressing shRNA-SHP-2 and shRNA-TC-PTP can beused in the methods and compositions described herein.

C. Beta Cell Specific Transcription

The third aspect in the CNIP approach is directed at the level of geneexpression and involves a transcriptional activator or transcriptionfactor, which is utilized as an attractor to converge and focus theglucose metabolism effect and metabolic/molecular effects generated byglucokinase, and the tyrosine kinase receptor(s) and tyrosine kinaseassociated receptor(s) to form beta cells in the adult pancreas. Theterm “transcription” refers to the process of copying a DNA sequence ofa gene into an RNA product, generally conducted by a DNA-directed RNApolymerase using the DNA as a template. Every system has a modulatorattractor, like in physics. In a chaotic system, the direction of thenetwork endpoint will follow the force of the attractor. From asimplistic view, the impact target of a projectile will depend on theinitial force of propulsion combined with air resistance and the effectof gravity on the projectile. The initial forces, air resistance andgravity, will act in synergy as an attractor to determine the finaldestination of the projectile. The living organism is a nonlineardynamic system that exists in a “chaotic” state. At the transcriptionallevel, the expression of a set of genes remains unchanged and thosegenes are call “housekeeping” genes. They carry out the routinefunctions of the cell, whereas other classes of genes are expressed inresponse to environmental stimuli. In the adaption of pancreatic betacells to a physiological stress, upregulation of gene expression isessential for the induction of pancreatic beta cell formation (Bouwensand Rooman, Physiol Rev 85:1255-1270, 2005). A key mediator of thisadaptive response to a physiological stress at the gene expression levelinvolves the activation of modular attractor(s) in the form oftranscription factors (Albert and Barabasi, Rev Mod Physics 74:47-97,2002; Albert and Othmer, J Theor Biol 223, 1-18, 2003). Therefore, theCNIP approach includes transcription factor(s) (as a modular attractor)that have been implicated in the formation of beta cells in the adultpancreas in response to physiological stress.

Pdx-1 overexpression alone could be used with a synergistic convergenceforce of other TFs to channel the CNIP gene expression pattern to inducepancreatic beta cell formation. Therefore, other TFs implicated in adultpancreatic beta cell formation and that can increase the effect of Pdx-1on beta cell formation in vivo can be used in the methods describedherein. In certain aspects, TFs implicated in beta cell formation can beused. TFs implicated in other endocrine cell formation can be excluded.The TFs implicated in pancreatic beta cell formation in thepost-development period added in combination with or in place of Pdx-1include: NeuroD, Isl1, Nkx6.1, and Pax4. Anti-diabetic compounds such asanti-diabetic thiazolidinediones (e.g., troglitazone) can also be usedin conjunction with TFs to increase beta cell formation. For example,troglitazone increases Pdx-1 expression in mouse islets through thefunctional peroxisome proliferators-activated receptor gamma (PPARγ)response element in the Pdx-1 promoter (Gupta et al., J Biol Chem.283(47):32462-70, 2008). Also contemplated is the induction of Pdx-1 viapositive modulation of the PPARγ response element in the promoter of thePdx-1 gene.

D. Therapeutic Compositions

In certain aspects, 1, 2, 3, or more of the therapeutic moietiesdescribed herein can be combined in one or more composition oradministered in combination. In one aspect, one or more therapeuticmoiety is provided as a cocktail of 1, 2, 3, or more nucleic deliveryvector(s) and/or therapeutic agent(s). Such a cocktail can beadministered orally, locally, or systemically as described herein.

In a further aspect, 2, 3, or more of the therapeutic moieties can bejoined to create a bi-valent, tri-valent, or tetra-valent composition.Such a composition can be administered orally, locally or systemicallyas described herein. In certain aspects, such compositions areadministered orally. In other aspects the compositions are injected orinfused locally.

In still a further aspect, 1, 2, 3, or more therapeutic moieties can bejoined in one molecule by chemical adaptor systems. Such a compositioncan be administered orally, locally or systemically as described herein.

II. NUCLEIC ACID COMPOSITIONS

The term “nucleic acid vector” is used to refer to a carrier nucleicacid molecule into which a nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated, transcribed, and/ortranslated (i.e., expressed). A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is “endogenous” to thecell but in a position within the host cell in which the sequence isordinarily not found. In certain aspects an exogenous vector can encodean endogenous nucleic acid. Nucleic acid vectors include plasmids,cosmids, viral genomes, and other expression vectors (bacteriophage,animal viruses, and plant viruses), artificial chromosomes (e.g., YACs),and the like. Given the current disclosure, one of skill in the artwould be well equipped to construct a vector through standardrecombinant techniques (see, for example, Maniatis et al., MolecularCloning: A laboratory Manual. Cold Spring Harbor Laboratory, New York,1989; Ausubel et al., Current Protocols in Molecular Biology, New YorkCity, N.Y., John Wiley & Sons, Inc., 1994, both incorporated herein byreference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for an RNA capable of beingtranscribed. In some cases, RNA molecules are then translated into aprotein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of inhibitory RNA,antisense molecules, or ribozymes. Expression vectors can contain avariety of “control sequences,” which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of an operablylinked coding sequence in a particular host cell. In addition to controlsequences that govern transcription and translation, vectors andexpression vectors may contain nucleic acid sequences that serve otherfunctions as well and are described herein.

Certain aspects involve the use of nucleic acids encoding beta cellinducing components. Examples of nucleic acids include GK as provided inSEQ ID NO:1 and SEQ ID NO:4; PTB1B shRNA as provided in SEQ ID NO:2; andPdx-1 as provided in SEQ ID NO:3; or the equivalent as would berecognized by one skilled in the art. In certain aspects the nucleicacid comprise a nucleotide sequence that is 80, 85, 90, 95, 98, or 100%identical to SEQ ID NO:1, 2, 3, and/or 4. In certain embodiments,nucleic acids of the invention encode proteins that are 80, 85, 90, 95,98, or 100% identical to the proteins of SEQ ID NO:5 (GK) or SEQ ID NO:6(Pdx-1) and maintain the appropriate activity.

The sequences may be modified, given the ability of several differentcodons to encode a single amino acid, while still encoding for the sameprotein or polypeptide. Optimization of codon selection can also beundertaken in light of the particular organism used for expression.

A. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. Additional promoter elements regulate thefrequency of transcriptional initiation. Typically, these are located inthe region 30-110 by upstream of the start site, although a number ofpromoters have been shown to contain functional elements downstream ofthe start site as well. To bring a coding sequence “under the controlof” a promoter, one positions the 5′ end of the transcription initiationsite of the transcriptional reading frame “downstream” of (i.e., 3′ of)the chosen promoter. The “upstream” promoter stimulates transcription ofthe DNA and promotes expression of the RNA. The spacing between promoterelements frequently is flexible, so that promoter function is preservedwhen elements are inverted or moved relative to one another. Dependingon the promoter, it appears that individual elements can function eithercooperatively or independently to activate transcription. A promoter mayor may not be used in conjunction with an “enhancer,” which refers to acis-acting regulatory sequence involved in the transcriptionalactivation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous” or “homologous.” Similarly, an enhancer maybe one naturally associated with a nucleic acid sequence, located eitherdownstream or upstream of that sequence. Alternatively, certainadvantages will be gained by positioning the nucleic acid under thecontrol of a recombinant, exogenous, or heterologous promoter, whichrefers to a promoter that is not normally associated with a nucleic acidsequence in its natural environment. A recombinant or heterologousenhancer refers also to an enhancer not normally associated with anucleic acid sequence in its natural environment. Such promoters orenhancers may include promoters or enhancers of other genes, andpromoters or enhancers isolated from another virus, or prokaryotic oreukaryotic cell, and promoters or enhancers not “naturally occurring,”i.e., containing different elements of different transcriptionalregulatory regions, and/or mutations that alter expression.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell, tissue, organ, or organism chosen for expression. Thoseof skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al., Molecular Cloning: A LaboratoryManual, vol. I. 2nd edition. Cold Spring Harbor Laboratory Press, 1989,incorporated herein by reference). The promoters employed may beconstitutive, tissue-specific, inducible, and/or useful under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The promoter may be heterologousor endogenous.

Additionally any promoter/enhancer combination (as per, for example, theEukaryotic Promoter Data Base EPDB, world-wide-web at epd.isb-sib.ch/)could also be used to drive expression. Use of a T3, T7, or SP6cytoplasmic expression system is another possible embodiment. Eukaryoticcells can support cytoplasmic transcription from certain bacterialpromoters if the appropriate bacterial polymerase is provided, either aspart of the delivery complex or as an additional genetic expressionconstruct.

In certain aspects, a nucleic acid of the invention can comprise anon-inducible or inducible promoter that will be expressed specificallyin the pancreatic tissues. Such non-inducible promoters includetissue-specific pancreas promoters from the insulin gene, glucagon gene,amylase gene, etc. Such inducible promoters include pancreas specificpromoters under the control of the glucose response element or pancreasspecific promoter under the control of a response element that isinducible by chemical, peptide, ligand, or metabolites.

B. Initiation Signals

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences.

Exogenous translational control signals, including the ATG initiationcodon, may need to be provided. One of ordinary skill in the art wouldreadily be capable of determining this and providing the necessarysignals. It is well known that the initiation codon must be “in-frame”with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. The exogenous translational controlsignals and initiation codons can be either natural or synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements.

C. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. “Restriction enzyme digestion” refers to catalyticcleavage of a nucleic acid molecule with an enzyme that function only atspecific locations in a nucleic acid molecule. Many of these restrictionenzymes are commercially available. Use of such enzymes is widelyunderstood by those of skill in the art. Frequently, a vector islinearized or fragmented using a restriction enzyme that cuts within theMCS to enable exogenous sequences to be ligated to the vector.“Ligation” refers to the process of forming phosphodiester bonds betweentwo nucleic acid fragments, which may or may not be contiguous with eachother. Techniques involving restriction enzymes and ligation reactionsare well known to those of skill in the art of recombinant technology.

D. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, the terminationsequences such as bovine growth hormone terminator or viral terminationsequences, such as the SV40 terminator. In certain embodiments, thetermination signal may be a lack of transcribable or translatablesequence, such as due to a sequence truncation.

E. Post-Transcriptional Regulatory Elements (PRE)

Post-transcriptional regulation is the control of gene expression at theRNA level, i.e., between the transcription and the translation of thegene. In certain aspects, the Woodchuck Hepatitis VirusPost-transcriptional Regulatory Element (WPRE) is used. WPRE increasesthe levels of nuclear transcripts and facilitates RNA export. WPRE mayfacilitate other steps in RNA processing, directing RNAs that wouldnormally be degraded within the nucleus to be efficiently expressed. TheWPRE can also function to facilitate the generation of RNA-proteincomplexes that would protect newly synthesized transcripts fromdegradation in the nucleus. (Zufferey et al., Journal of Virology, 73:2886-2892, 1999 and U.S. Pat. No. 6,284,469, which is incorporatedherein by reference).

F. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal or the bovine growth hormone polyadenylationsignal, convenient and known to function well in various target cells.Polyadenylation may increase the stability of the transcript or mayfacilitate cytoplasmic transport.

G. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

H. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with fluorescence assisted cell sorting (FACS) and/orimmunohistochemistry. The marker used is not believed to be important,so long as it is capable of being expressed simultaneously with thenucleic acid encoding a gene product. Further examples of selectable andscreenable markers are well known to one of skill in the art.

III. POLYPEPTIDE COMPOSITIONS

Modifications and/or changes may be made in the amino acid compositionof polypeptides, and thus the present invention contemplates variationin sequences of the polypeptides, and nucleic acids coding therefor,where they are nonetheless able retain substantial activity with respectto the therapeutic, preventative, and curative aspects of the presentinvention.

The biological functional equivalent may comprise a polynucleotide thathas been engineered to contain distinct sequences while at the same timeretaining the capacity to encode the “wild-type” or standard peptide.This can be accomplished through the degeneracy of the genetic code,i.e., the presence of multiple codons, which encode for the same aminoacids. In one example, one of skill in the art may wish to introduce arestriction enzyme recognition sequence into a polynucleotide while notdisturbing the ability of that polynucleotide to encode a protein.

In another example, a polynucleotide may encode a biological functionalequivalent with more significant changes. Certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies, binding sites onsubstrate molecules, receptors, and such like. So-called “conservative”changes do not disrupt the biological activity of the protein, as thestructural change is not one that impinges on the protein's ability tocarry out its designed function. It is thus contemplated by theinventors that various changes may be made in the sequence of genes andproteins disclosed herein, while still fulfilling the goals of thepresent invention.

In terms of functional equivalents, it is well understood by the skilledartisan that, inherent in the definition of a “biologically functionalequivalent” protein and/or polynucleotide, is the concept that there isa limit to the number of changes that may be made within a definedportion of the molecule while retaining a molecule with an acceptablelevel of equivalent biological activity. Biologically functionalequivalents are thus defined herein as those proteins (andpolynucleotides) in selected amino acids (or nucleotides) may besubstituted. In certain aspects, a polypeptide is 80, 85, 90, 92, 94,96, 98, or 100% identical to the wildtype form of the polypeptide. Incertain aspects, polypeptide(s) 80, 85, 90, 92, 94, 96, 98, or 100%identical to SEQ ID NO: 5 or 6 are used or nucleic acids encoding thesame.

In general, the shorter the length of the molecule, the fewer changesthat can be made within the molecule while retaining function. Longerdomains may have an intermediate number of changes. The full-lengthprotein will have the most tolerance for a larger number of changes.However, it must be appreciated that certain molecules or domains thatare highly dependent upon their structure may tolerate little or nomodification. Function of a polypeptide can be determined by usingvarious assays know to detect the activity of the polypeptide ofinterest.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and/or the like. Ananalysis of the size, shape and/or type of the amino acid side-chainsubstituents reveals that arginine, lysine, and/or histidine are allpositively charged residues; that alanine, glycine, and/or serine areall a similar size; and/or that phenylalanine, tryptophan, and/ortyrosine all have a generally similar shape. Therefore, based upon theseconsiderations, arginine, lysine, and/or histidine; alanine, glycine,and/or serine; and/or phenylalanine, tryptophan, and/or tyrosine aredefined herein as biologically functional equivalents.

To effect more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and/or chargecharacteristics, these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and/or arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index and/or score and/or stillretain a similar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those that are within ±1 areparticularly preferred, and/or those within ±0.5 are even moreparticularly preferred.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. Asdetailed in U.S. Pat. No. 4,554,101, the following hydrophilicity valueshave been assigned to amino acid residues: arginine (+3.0); lysine(+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). In makingchanges based upon similar hydrophilicity values, the substitution ofamino acids whose hydrophilicity values are within ±2 is preferred,those that are within ±1 are particularly preferred, and/or those within±0.5 are even more particularly preferred.

In certain embodiments recombinant polypeptides as described hereincomprise protein transduction domains. Protein transduction domains(PTDs) have the ability to translocate across biological membranes. ThePTDs are relatively short (one- to 35-amino acid) sequences that conferthis apparent translocation activity to proteins and othermacromolecular cargo to which they are conjugated, complexed or fused.The HIV-derived TAT peptide (YGRKKRRQRRR (SEQ ID NO:7)), for example,has been used widely for intracellular delivery of various agentsranging from small molecules to proteins, peptides, range ofpharmaceutical nanocarriers and imaging agents. Alternatively,receptor-mediated endocytic mechanisms can also be used forintracellular drug delivery. For example, the transferrinreceptor-mediated internalization pathway is an efficient cellularuptake pathway that has been exploited for site-specific delivery ofdrugs and proteins. This is achieved either chemically by conjugation oftransferrin with therapeutic drugs or proteins or genetically byinfusion of therapeutic peptides or proteins into the structure oftransferrin. Naturally existing proteins (such as the iron-bindingprotein transferrin) are very useful in this area of drug targetingsince these proteins are biodegradable, nontoxic, and non-immunogenic.Protein transduction domains include, but are not limited to, PTDsderived from proteins such as human immunodeficiency virus 1 (HIV-1) TAT(Ruben et al., J. Virol. 63:1-8, 1989), e.g., GRKKRRQRRR (TAT 48-57, SEQID NO:8); the herpes virus tegument protein VP22 (Elliott and O'HareCell 88:223-33, 1997); the homeotic protein of Drosophila melanogasterAntennapedia (Antp) protein (Penetratin PTD; Derossi et al. J. Biol.Chem. 271:18188-93, 1996); the protegrin 1 (PG-1) anti-microbial peptideSynB (e.g., SynB1, SynB3, and Syn B4; Kokryakov et al. FEBS Lett.327:231-36, 1993); and basic fibroblast growth factor (Jans FASEB J.8:841-47, 1994). PTDs also include synthetic PTDs, such as, but notlimited to, polyarginine peptides (Futaki et al., J. Mol. Recognit.16:260-64, 2003; Suzuki et al., J. Biol. Chem. 276:5836-40, 2001);transportan (Pooga et al., FASEBJ. 12:67-77, 1988; Pooga et al., FASEBJ. 15:1451-53, 2001); MAP (Oehlke et al., Biochim. Biophys. Acta.1414:127-39, 1998); KALA (Wyman et al., Biochemistry 36:3008-17, 1997);and other cationic peptides, such as, for example, various β-cationicpeptides (Akkarawongsa et al., Antimicrob. Agents and Chemother.52(6):2120-29, 2008).

IV. DELIVERY VECTORS

In certain aspects, components are provided to a pancreas or other organor tissue by using nucleic acids that encode or express such components.Viral and non-viral delivery vectors can be used in the methodsdescribed herein (e.g., Syner-III). The term “nucleic acids”, “nucleicacid molecules”, “nucleic acid sequences”, “nucleotide sequences” and“nucleotide molecules” are used interchangeably herein and, unlessotherwise specified, refer to a polymer of deoxyribonucleic acids,including cDNA, DNA, PNA, or polymers of ribonucleic acids (RNA).Nucleic acid may be obtained from a cellular extract, genomic orextragenomic DNA, viral nucleic acids, or artificially/chemicallysynthesized molecules. The term can include double stranded or singlestranded deoxyribonucleic or ribonucleic acids.

A. Viral Delivery

The ability of certain viruses to infect cells or enter cells viareceptor-mediated endocytosis, and to express virally encoded genes havemade them attractive candidates for the transfer of foreign nucleicacids into cells (e.g., mammalian cells). Viruses may thus be utilizedthat encode and express agents to increase the activity of glucosemetabolism, increase tyrosine kinase receptor activity, and increasetranscription of genes associated with beta cells. Non-limiting examplesof virus vectors that may be used to deliver nucleic acids are describedbelow.

Adenoviral Vectors.

A particular method for delivery of the nucleic acid involves the use ofan adenovirus expression vector. Although adenovirus vectors are knownto have a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a tissue orcell-specific construct that has been cloned therein. Knowledge of thegenetic organization or adenovirus, a 36 kb, linear, double-stranded DNAvirus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus and Horwitz, Semin. Virol. 3,237-252, 1992).

AAV Vectors.

The nucleic acid may be introduced into the cell usingadenovirus-assisted transfection. Increased transfection efficiencieshave been reported in cell systems using adenovirus-coupled systems.Adeno-associated virus (AAV) has a low frequency of integration and itcan infect non-dividing cells, thus making it useful for delivery ofgenes into mammalian cells in tissue culture or in vivo. AAV has a broadhost range for infectivity. Details concerning the generation and use ofrAAV vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797,368,each of which incorporated herein by reference.

Retroviral Vectors.

Retroviruses have the ability to integrate their genes into the hostgenome, transferring a large amount of foreign genetic material,infecting a broad spectrum of species and cell types and of beingpackaged in special cell-lines. In order to construct a retroviralvector, a nucleic acid (e.g., one encoding a protein of interest) isinserted into the viral genome in the place of certain viral sequencesto produce a virus that is replication-defective. In order to producevirions, a packaging cell line containing the gag, pol, and env genesbut without the LTR and packaging components is constructed. When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into a special cell line (e.g., bycalcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media. Themedia containing the recombinant retroviruses is then collected,optionally concentrated, and used for gene transfer. Retroviral vectorsare able to infect a broad variety of cell types.

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, U.S. Pat. Nos. 6,013,516 and 5,994,136, each of whichis incorporated herein by reference). Some examples of lentivirusinclude the Human Immunodeficiency Viruses: HIV-1, HIV-2 and the SimianImmunodeficiency Virus: SIV. Lentiviral vectors have been generated bymultiply attenuating the HIV virulence genes, for example, the genesenv, vif, vpr, vpu and nef are deleted making the vector biologicallysafe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat is describedin U.S. Pat. No. 5,994,136, which is incorporated herein by reference.

One may target the recombinant virus by linkage of an envelope proteinwith an antibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene thatencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific. Such viral vectors can betargeted to cells of the pancreas.

Other Viral Vectors.

Other viral vectors may be employed in the methods of the presentinvention. Vectors derived from viruses such as vaccinia virus(Ridgeway, In: Vectors: A survey of molecular cloning vectors and theiruses, Rodriguez and Denhardt (Eds.), Stoneham: Butterworth, 467-492,1988; Baichwal and Sugden, In: Gene Transfer, Kucherlapati (Ed.), NY,Plenum Press, 117-148, 1986; Coupar et al., Gene 68:1-10, 1988), sindbisvirus, cytomegalovirus, and herpes simplex virus may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, Science, 244:1275-1281, 1989; Ridgeway, In: Vectors: Asurvey of molecular cloning vectors and their uses, Rodriguez andDenhardt (Eds.), Stoneham: Butterworth, 467-492, 1988; Baichwal andSugden, In: Gene Transfer, Kucherlapati (Ed.), NY, Plenum Press,117-148, 1986; Coupar et al., Gene 68:1-10, 1988; Horwich et al., J.Virol. 64:642-650, 1990).

Modified Viruses.

A nucleic acid to be delivered may be housed within an infective virusthat has been engineered to express or is coupled to a specific bindingligand. The virus particle will thus bind specifically to the cognatereceptors of the target cell and deliver the contents to the cell. Anovel approach designed to allow specific targeting of virus vectors wasdeveloped based on the chemical or genetic modification of a virus bythe chemical addition or recombinant engineering of moieties to or insurface proteins of the virus. One such modification using lactosemoieties can permit the specific infection of hepatocytes viasialoglycoprotein receptors.

Another approach to targeting of recombinant viruses was designed inwhich biotinylated antibodies against a viral surface protein andagainst a specific cell receptor were used. The antibodies can becoupled via biotin by using streptavidin (Roux et al., Proc. Natl. Acad.Sci. USA, 86:9079-83, 1989). Using antibodies against majorhistocompatibility complex class I and class II antigens, theydemonstrated the infection of a variety of human cells that bore thosesurface antigens with an ecotropic virus in vitro (Roux et al., Proc.Natl. Acad. Sci. USA, 86:9079-83, 1989).

In certain aspects, an adaptor system can be used, i.e., a molecule thatbinds both the delivery vector and a target-pancreatic cell receptor tofacilitate transduction in the pancreas. In a further aspect, use of anative viral vector receptor can be fused to the pancreas targetingligand. In still another aspect, a bispecific antibody, two antibodiescoupled together, can be coupled to the delivery vector resulting thedelivery vector having specificity for the target pancreatic cells. Incertain aspects, a targeting moiety can be bound to the delivery vectorby chemical means. In further aspects, an antibody that binds to agenetically incorporated Ig-binding domain of the delivery vector can beused to enhance delivery. In further aspects, small targeting motifs canbe inserted into the capsid, envelope, viral attachment, other viralsurface protein to target the pancreatic cells.

In certain aspects, a viral construct can encode for two heterologousprotein components (e.g., GK and Pdx-1) and express an shRNA PTP1B. In afurther aspect, each component can be comprised in individual andseparate viruses.

The compositions described herein can be delivered via the pancreaticduct through endoscopy. Briefly, the patient is sedated oranaesthetized, and a flexible endoscope is inserted through the mouth,down the esophagus, into the stomach, through the pylorus into theduodenum where the ampulla of Vater (the opening of the common bile ductand pancreatic duct) exists. The sphincter of Oddi is a muscular valvethat controls the opening of the ampulla. The region can be directlyvisualized with the endoscopic camera while performing the procedure. Aplastic catheter or cannula is inserted through the ampulla, and thebeta cell inducing composition (e.g., Syner-III) is introduced into thepancreatic bile duct to target the pancreas. The beta cell formingcomposition can also be delivered intravenously by coupling a viralvector with a pancreas targeting moiety, e.g., modifying the envelopstructure of viral vector to target only the pancreatic tissues.

B. Lipid-Mediated Transfection

In a further embodiment, a nucleic acid may be entrapped in a lipidparticle such as, for example, a liposome. Liposomes are vesicularstructures characterized by a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, In: Liver Diseases, Targeted Diagnosis and Therapy UsingSpecific Receptors and Ligands, Wu et al. (Eds.), Marcel Dekker, NY,87-104, 1991). Also contemplated is a nucleic acid complexed withLipofectamine (Gibco BRL) or Superfect (Qiagen).

Lipid-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful (Nicolau and Sene, Biochim. Biophys.Acta, 721:185-190, 1982; Fraley et al., Proc. Natl. Acad. Sci. USA,76:3348-3352, 1979; Nicolau et al., Methods Enzymol. 149:157-176, 1987).The feasibility of lipid-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells has also beendemonstrated (Wong et al., Gene 10:87-94 1980).

In certain embodiments of the invention, a lipid particle may becomplexed with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry oflipid-encapsulated DNA (Kaneda et al., Science 243:375-378 1989). Inother embodiments, a lipid particle may be complexed or employed inconjunction with nuclear non-histone chromosomal proteins (HMG-1) (Katoet al., J. Biol. Chem. 266:3361-3364, 1991). In yet further embodiments,a lipid particle may be complexed or employed in conjunction with bothHVJ and HMG-1. In other embodiments, a delivery vehicle may comprise aligand and a lipid particle.

In certain aspects, components described herein, including shRNA ofPTP1B and nucleic acids encoding Pdx-1 and glucokinase can be deliveredby liposomes. Liposomes containing the nucleic acids could be deliveredintravenously and liberated when it reaches the pancreatic tissues. Incertain aspects, nucleic acids of the invention are incorporated intoliposomes that contain chemically coupled ligands that are presented onthe liposome surface. The ligands specifically target pancreatic cells.Using this strategy, a variety of ligands or receptors, such asantibodies, growth factors, cytokines, hormones, and toxins, can beanchored on liposome surface so that the beta cell inducing componentscan be targeted to and introduced into pancreatic cells.

C. Receptor Mediated Transfection

Still further, a nucleic acid may be delivered to a target cell viareceptor-mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis thatwill be occurring in a target cell. In view of the cell type-specificdistribution of various receptors, this delivery method adds anotherdegree of specificity to the present invention.

Certain receptor-mediated gene targeting vehicles comprise a cellreceptor-specific ligand and a nucleic acid-binding agent. Otherscomprise a cell receptor-specific ligand to which the nucleic acid to bedelivered has been operatively attached. Several ligands have been usedfor receptor-mediated gene transfer (Wu and Wu, J. Biol. Chem.262:4429-4432, 1987; EP patent 0273085), which establishes theoperability of the technique. In certain aspects of the presentinvention, a ligand will be chosen to correspond to a receptorspecifically expressed on the target cell population.

In other embodiments, a nucleic acid delivery vehicle component of acell-specific nucleic acid targeting vehicle may comprise a specificbinding ligand in combination with a lipid particle. The nucleic acid(s)to be delivered are housed within the lipid particle and the specificbinding ligand is functionally incorporated into the lipid layer. Thelipid particle will thus specifically bind to the receptor(s) of atarget cell and deliver the contents to a cell. Such systems have beenshown to be functional using systems in which, for example, epidermalgrowth factor (EGF) is used in the receptor-mediated delivery of anucleic acid to cells that exhibit upregulation of the EGF receptor.

In still further embodiments, the nucleic acid delivery vehiclecomponent of a targeted delivery vehicle may be a lipid particle itself,which will preferably comprise one or more lipids or glycoproteins thatdirect cell-specific binding. For example, lactosyl-ceramide, agalactose-terminal asialganglioside, have been incorporated into lipidparticles and observed to increase the uptake of the insulin gene byhepatocytes (Nicolau et al., Methods Enzymol. 149:157-176, 1987). It iscontemplated that the tissue-specific transforming constructs of thepresent invention can be specifically delivered into a target cell in asimilar manner or as described herein.

D. Microprojectile Bombardment

Microprojectile bombardment techniques can be used to introduce anucleic acid into at least one, organelle, cell, tissue or organism(U.S. Pat. Nos. 5,550,318; 5,538,880; 5,610,042; and PCT Application WO94/09699; each of which is incorporated herein by reference). Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., Nature 327, 70-73, 1987).There are a wide variety of microprojectile bombardment techniques knownin the art, many of which are applicable to the invention.

In this microprojectile bombardment, one or more particles may be coatedwith at least one nucleic acid and delivered into cells by a propellingforce. Several devices for accelerating small particles have beendeveloped. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force.The microprojectiles used have consisted of biologically inertsubstances such as tungsten or gold particles or beads. Exemplaryparticles include those comprised of tungsten, platinum, and gold. It iscontemplated that in some instances DNA precipitation onto metalparticles would not be necessary for DNA delivery to a recipient cellusing microprojectile bombardment. However, it is contemplated thatparticles may contain DNA rather than be coated with DNA. DNA-coatedparticles may increase the level of DNA delivery via particlebombardment but are not, in and of themselves, necessary.

Naked plasmid DNA can be transferred into a cell by air pistol (GeneGun). Syner-III plasmid could be injected via the pancreatic duct usingthe gene gun approach with endoscopy to reach the pancreatic tissues.The plasmid DNA bombardments through a gene gun enter the cell byphysical pressure that opens membrane pores and/or by facilitatingdiffusion of the naked plasmid DNA though the cell membrane.

E. Nanoparticles

Nucleic acids of the invention can be incorporated intothree-dimensional, multicomponent structures of nanoparticles thattarget pancreatic or other tissues. The nanoparticles used include, butare not limited to liposomes, polymers, proteins, micelles, dendimers,quantum dots, nanoshells, nanocyrstals, gold nanoparticles, paramagneticnanoparticles, and carbon nanotubes.

F. Hydrodynamic Gene Delivery

Naked plasmid DNA can be delivered via the pancreatic duct employinghydrodynamic gene delivery. A balloon catheter can be placed in thepancreatic duct. The balloon catheter placed in the pancreatic duct isinflated for occlusion-assisted infusion.

G. Electroporation

In certain aspects, a pair of electrodes can be placed on or inpancreatic or other tissues, and nucleic acids of the invention aredeposited on the electrodes so that the genetic material is transferredinto the tissues.

H. Ultrasound-Facilitated Gene Transfer

Nucleic acids of the invention can be incorporated into microbubblesthat are injected intravenously. When the microbubbles reach thepancreas or other tissue they are targeted with ultrasound. As themicorbubbles expand and burst, they release the nucleic acids in thepancreas. The local shock waves cause nucleic acids to permeate thenearby cell membranes.

I. Gene Transfer by Needle

In certain aspects, nucleic acids of the invention are injected directlyinto the pancreas or other tissue using a needle.

V. PHARMACEUTICAL COMPOSITIONS

In light of the current specification, the determination of anappropriate treatment regimen (e.g., dosage, frequency ofadministration, systemic vs. local, etc.) is within the skill of theart. For administration, the components described herein will beformulated in a unit dosage form (solution, suspension, emulsion, etc.)in association with a pharmaceutically acceptable carrier. Such vehiclesare usually nontoxic and non-therapeutic. Examples of such vehicles arewater, saline, Ringer's solution, dextrose solution, and Hank'ssolution. Non-aqueous vehicles such as fixed oils and ethyl oleate mayalso be used. A preferred vehicle is 5% (w/w) human albumin in saline.The vehicle may contain minor amounts of additives, such as substancesthat enhance isotonicity and chemical stability, e.g., buffers andpreservatives.

The therapeutic compositions described herein, as well as theirbiological equivalents, can be administered independently or incombination by any suitable route. Examples of parenteral administrationinclude intravenous, intraarterial, intramuscular, intraperitoneal, andthe like. The routes of administration described herein are merely anexample and in no way limiting.

The dose of the therapeutic compositions administered to an animal,particularly in a human, in accordance with embodiments of theinvention, should be sufficient to result in a desired response in thesubject over a reasonable time frame. It is known that the dosage oftherapeutic compositions depends upon a variety of factors, includingthe strength of the particular therapeutic composition employed, theage, species, condition or disease state, and the body weight of theanimal.

Moreover, dose and dosage regimen, will depend mainly on the type ofbiological damage to the host, the type of subject, the history of thesubject, and the type of therapeutic composition being administered. Thesize of the dose will be determined by the route, timing and frequencyof administration as well as the existence, nature and extent of anyadverse side effects that might accompany the administration of aparticular therapeutic composition and the desired physiological effect.It is also known that various conditions or disease states, inparticular, chronic conditions or disease states, may require prolongedtreatment involving multiple administrations.

Therefore, the amount of the therapeutic composition must be effectiveto achieve an enhanced therapeutic index. If multiple doses areemployed, the frequency of administration will depend, for example, onthe type of subject. One skilled in the art can ascertain upon routineexperimentation the appropriate route and frequency of administration ina given subject that are most effective in any particular case. Suitabledoses and dosage regimens can be determined by conventionally knownrange-finding techniques. Generally, treatment is initiated with smallerdosages, which are less than the optimal dose of the compound.Thereafter, the dosage is increased by small increments until theoptimal effect under the circumstances is obtained.

The therapeutic compositions for use in embodiments of the inventiongenerally include carriers. These carriers may be any of thoseconventionally used and are limited only by the route of administrationand chemical and physical considerations, such as solubility andreactivity with the therapeutic agent. In addition, the therapeuticcomposition may be formulated as polymeric compositions, inclusioncomplexes, such as cyclodextrin inclusion complexes, liposomes,microspheres, microcapsules, and the like, without limitation.

The pharmaceutically acceptable excipients described herein, forexample, vehicles, adjuvants, carriers, or diluents, are well known andreadily available. It is preferred that the pharmaceutically acceptablecarrier be one which is chemically inert with respect to the therapeuticcomposition and one that has no detrimental side effects or toxicityunder the conditions of use.

The choice of excipient will be determined, in part, by the particulartherapeutic composition, as well as by the particular method used toadminister the composition. Accordingly, there are a wide variety ofsuitable formulations of the pharmaceutical composition used in theembodiments of the invention. For example, the non-limiting formulationscan be injectable formulations such as, but not limited to, those forintravenous, subcutaneous, intramuscular, intraperitoneal injection, andthe like, and oral formulations such as, but not limited to, liquidsolutions, including suspensions and emulsions, capsules, sachets,tablets, lozenges, and the like. Non-limiting formulations suitable forparenteral administration include aqueous and non-aqueous isotonicsterile injection solutions, including non-active ingredients such asantioxidants, buffers, bacteriostats, solubilizers, thickening agents,stabilizers, preservatives, surfactants, and the like. The solutions caninclude oils, fatty acids, including detergents and the like, as well asother well known and common ingredients in such compositions, withoutlimitation.

VI. EXAMPLES

The following examples as well as the figures are included todemonstrate certain embodiments of the invention. It should beappreciated by those of skill in the art that the techniques disclosedin the examples or figures represent techniques discovered by theinventors to function well in the practice of the described methods, andthus can be considered to constitute modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

Example 1 Beta Cell Formation in Adult Pancreas

A. Results

For illustration purposes, the therapeutic moieties includeLentivirus-CMV-Glucokinase (GK), Lentivirus-H1-shRNA PTP1B; andLentivirus-CMV-PDX-1. The control composition includesLentivirus-CMV-GFP and Lentivirus-H1-shRNA Scramble injected at the sameconcentration as the therapeutic composition. Four-weeks post-injectionin vivo over-expression of PDX-1 and GK, and suppression of PTP1Bexpression was detected in the mouse pancreas. Glucokinaseover-expression was detected in the islets and in exocrine tissues.Expression of glucokinase in the exocrine tissues confirms theover-expression of the glucokinase by the therapeutic composition sincepancreatic tissues only express glucokinase in endocrine cells. Pdx-1over expression is detected by using a c-Myc tag incorporated into thecDNA of Pdx-1 to differentiate between exogenous and endogenousexpression. shRNA PTP1B co-expressing GFP was also detected. Proteinexpression was confirmed by western blot.

First Aspect of CNIP Approach:

One goal of the CNIP approach is to increase glucose metabolism in thepancreas to induce pancreatic beta cell formation. For glucose to enterthe glycolytic pathway, it first must enter the intracellular spacethrough a membrane glucose transport system. The glucose transporters,Glut 1 and Glut 3, are present in exocrine and endocrine humanpancreatic tissues, and Glut 2 is present in the pancreatic beta cell(Coppieters et al., Diabetes Metab Res Rev 27: 746-754, 2011). However,glucose transport is not the rate-limiting step for glucose entry intothe glycolytic pathway (Wasserman et al., J. Exp. Biol. 214:254-262,2011). The first rate-limiting step for glucose metabolism in theglycolytic pathway is at the level of glucose phosphorylation byhexokinase. The phosphorylation of glucose by hexokinase produces themetabolite glucose 6-phosphate (G-6-P). The pancreatic beta cellscontain a hexokinase type IV, named glucokinase (GK). Glucokinase is notallosterically inhibited by the accumulation of intracellular G-6-P, incontrast to other hexokinases. Therefore, for glucokinase, the amountand activity of the glucokinase enzyme regulates the rate of glucoseflux through glycolysis. For other members of the hexokinase family,product inhibition by G-6-P is the key regulator of enzyme activity and,therefore, glucose entry into the glycolytic pathway. If glucose is notphosphorylated by hexokinase, it cannot undergo further metabolism andcannot generate any signal to the transcriptional machinery to inducegene expression (Doiron et al., J Biol Chem. 269: 10213-10216, 1994 andJ Biol Chem. 271:5321-5324, 1996). Therefore, in certain aspectsglucokinase can be included in a CNIP cocktail to induce pancreatic betacell formation.

The inventors designed a lentiviral vector construct expressing theglucokinase gene under control of the cytomegalovirus (CMV) promoter(FIG. 2A). The lentiviral vector construct included aposttranscriptional regulatory element of woodchuck hepatitis virus(WPRE) at the 3′ untranslated region of coding sequence, which increasedthe level of expression of the transgene. WPRE functions within thenucleus to stimulate gene expression posttranscriptionally by increasingthe levels of nuclear transcripts and greatly increasing the RNAhalf-life (Zufferrey et al., J. Virol. 73:2886-289, 1999). The mouseglucokinase (GCK) gene was subcloned to pEntCMV-WPRE vector and theinsert was verified by DNA sequencing. The pENT-GCK was treated with LRClonase II enzyme (Invitrogen) and ligated to a pLenti vector. Therecombination product was transformed into E. coli cells. Afterovernight incubation, the positive clones were selected, and plasmid DNAwas purified. The pEnt-GCK and pLenti-GCK were transfected into 293cells. 48 hours after transfection, the cells were lysed in SDS-PAGEbuffer and subjected to 4-20% SDS-PAGE gel electrophoresis and analyzedby Western blotting. The Western blot was carried out using the anti-GCKantibody at a 1:1000 dilution, followed by a HRP conjugated secondaryantibody. The Western blot membrane was developed using ECL reagents.

The pLenti-GCK is used for the production of pure, high titer lentiviralvector. The Lenti-GCK will be injected directly into the pancreas ofadult mice as described above. Adult mice C57BL/6 (8 weeks old; fromCharles River, Wilmington, Mass.) will be used for the in vivoexperiment targeting the pancreas with lentiviral vector construct.

Increased glucokinase expression with a plasmid vector has been shown toincrease the glucose-6-phosphate (G-6-P) pool (Doiron et al., J BiolChem. 269: 10213-10216, 1994). Glucose 6-phosphate is a key intermediatethat sits at the junction of several metabolic pathways (glycolysis,gluconeogenesis, pentose phosphate pathway, glycogenesis andglycogenolysis). Doiron et al (1996) demonstrated that the glucosesignal to the transcriptional machinery is mediated by xylulose5-phosphate, which is a metabolite produced by the pentose phosphatepathway. As demonstrated by Doiron, xylulose 5-phosphate is the majormetabolite responsible for mediating transcriptional machinery inductionby glucose metabolism. The key enzyme that regulates metabolic fluxthrough the pentose phosphate pathway is glucose-6-phosphatedehydrogenase (G6PD). G-6-P enters the pentose phosphate pathway throughthe action of G6PD enzyme. Therefore, in the development of our CNIPapproach, the combination of Lenti-CGK and Lenti-G6PD to channel glucose6-phosphate into the pentose phosphate pathway to enhancexylulose-5-phosphate formation that mediates the transcriptional effectsof glucose metabolism can be used. Another important enzyme in theformation of xylulose 5-phosphate is transketolase (TK), which islocated further down the pentose phosphate pathway. Therefore, analternative approach will be to combine Lenti-TK with Lenti-CGK toincrease the effect of glucose metabolism regulated gene expression. Thesynergistic action of all molecules (Lenti-CGK, Lenti-G6PD and Lenti-TK)can be used to activate the transcriptional machinery under the controlof glucose metabolism and increase beta cell formation.

Second Aspect of CNIP Approach:

The second aspect of the CNIP approach includes increasing membranereceptor tyrosine kinase activity and tyrosine kinase associatedreceptor activity. Glucose metabolism is a major mechanism involved inthe formation of beta cells in the adult pancreas. Another systemimplicated in beta cell formation is ligand binding to tyrosine kinasereceptor(s). (Vasavada et al., Int J Biochem Cell Biol. 38:931-950,2006). The increase in pancreatic beta cell mass in response tophysiological stress (i.e. pregnancy) is mediated by growth hormone,prolactin, and placental lactogen working through the prolactinreceptor. The prolactin receptor does not have intrinsic tyrosine kinaseactivity but it interacts with members of the Janus kinase family oftyrosine kinases. Prolactin receptor is part of the family of tyrosinekinase associated receptor(s). The Janus kinase family of tyrosinekinases is responsible for the increase in beta cell mass in response tohormonal changes that occur in pregnancy (Vasavada et al., Int J BiochemCell Biol. 38:931-950, 2006). The action of insulin, insulin-like growthfactors, hepatocyte growth factor, and epidermal growth factor alsoincrease beta cell mass by activating a membrane bound tyrosine kinasereceptor (Vasavada et al., Int J Biochem Cell Biol. 38:931-950, 2006).Therefore, lactogens (including growth hormone, prolactin and placentallactogen), insulin, insulin-like factors, hepatocyte growth factor andepidermal growth factor receptor require the activation of tyrosinekinase(s) to increase pancreatic beta cells mass.

Protein-tyrosine phosphatase 1B (PTP1B) has been shown to inhibit theability of insulin, insulin-like growth factors receptor, prolactin andhepatocyte growth factor to activate tyrosine kinase(s) (Aoki andMatsuda, J. Biol. Chem. 275:39718-39726, 2000; Kakazu et al., InvestOpthalmol Vis Sci. 49:2927-2935, 2008; Tonks, Nat Rev. Mol Cell Biol7:833-46, 2006). Consequently, one molecule that can be included in aCNIP cocktail is an inhibitor or inhibitory RNA (e.g., shRNA) targetingPTP1B. The suppression of PTP1B protein production by shRNA willincrease receptor tyrosine kinase activity involved in beta cellformation in the adult pancreas in response to binding of aphysiological concentration of the corresponding hormone. The shRNAPTP1B has been introduced into a lentivirus construct using the samemethod described above for lenti-CGK under the control of the H1polymerase promoter (Doiron et al., Diabetologia 55:719-728, 2012) (FIG.2C). The polymerase III promoter H1 is active ubiquitously in all cells,because of the housekeeping function of polymerase III. Thelenti-shRNA-PTP1B was provided to the pancreas as described above.

Lentivirus shRNA PTP1B. A small hairpin RNAi was constructed into pEQU6vector. The target sequences are 20 nt in length (GCCAGGACATTCGACATGAASEQ ID NO:2). The shRNAi sequences were verified by DNA sequencing.

Co-transfection experiments were performed using the target geneexpression plasmid pEnt-PTP1B and one pEQ-PTP1B-shRNA vector. The chartbelow describes the component of each reaction. 48 hours aftertransfection, cells were lysed in SDS-PAGE buffer and subjected to 4-20%SDS-PAGE gel electrophoresis and Western blot analyses. The Western blotwas carried out using the anti-PTP1B antibody at a 1:1000 dilution. Themembrane was evaluated using ECL reagents. For each 6-well:

293 cells PTP1B-shRNA mouse PTP1B cDNA 1.0 pEQ- PTP1B shRNA 2.0pEQ-scramble-shRNA 3.0 Total DNA 3.0 3.0

Third Aspect of CNIP Approach:

A third aspect that can be included in the methods described hereinincludes increasing gene expression through transcriptional factor(s) tointegrate the effect of glucose metabolism (Aspect 1) and stimulation oftyrosine kinase receptor family activity (Aspect 2) to induce beta cellformation in the pancreas.

Different models have been proposed to explain the facilitated diffusionof transcriptional factors to bind to their target DNA sequence into thenucleus. One model proposes that the transcriptional factor (TF) bindsthe DNA by facilitated diffusion. First, the TF interacts with the DNArandomly at a non-specific site. After initial TF interaction with theDNA molecules, by facilitated diffusion, the TF moves from its initialnon-specific site to its target sequence by ‘sliding’ along the DNA. Asthe TF rolls along the DNA and finds its corresponding binding site, itinduces activation or inhibition of the transcriptional machinery.Irrespective of the model proposed to explain how the TF reaches its DNAbinding site, all proposed models and experiments include facilitateddiffusion with random movement of the TF to find its site of activationor inhibition in the transcriptional machinery. One action of glucosemetabolism signaling to the transcriptional machinery is to increase theprobability of the TF binding the DNA molecule (Doiron et al., J BiolChem. 271:5321-5324, 1996). Indeed, glucose metabolism signaling to thetranscriptional machinery induces TF expression and TF translocationfrom the cytoplasm to the nucleus turns on glucose-induced genes (Doironet al., J Biol Chem. 271:5321-5324, 1996). Consequently, the biophysicalmechanism by which TFs increase glucose-induced gene expression dependson the TF quantity level present in the nucleus. Indeed, when thequantity of TFs in the nucleus increases, the chances of interactingwith DNA molecules to randomly find its specific binding site increased(Mitanchez et al., Endo Rev 18:520-540, 1997). Increasing expression ofgenes implicated in beta cell formation in the adult pancreas can beaccomplished or enhanced by overexpressing key TFs that activate thebeta cell formation pathway.

One of the major transcriptional factors implicated in beta cellformation after birth is Pdx-1 (Doiron and DeFronzo, Int J EndocrinolMetab. 9:356-357, 2011). Pdx-1 has distinct effects before and afterbirth in the pancreas (Doiron and DeFronzo, Int J Endocrinol Metab.9:356-357, 2011). At the embryonic stage, Pdx-1 is essential forpancreatic development and it is expressed in endocrine and exocrinetissues. However, after birth, Pdx-1 is expressed only in pancreaticbeta cells and somatostatin cells. Therefore, in the adult pancreas,Pdx-1 does not induce embryonic pancreatic development but it doesinduce pancreatic beta cell formation by its action to induce insulingene expression in response to glucose metabolism signaling to thetranscriptional machinery (Doiron and DeFronzo, Int J Endocrinol Metab.9:356-357, 2011; Mitanchez et al., Endo Rev 18:520-540, 1997). Pdx-1 hasbeen demonstrated in post-development to induce beta cell formation inadult animals (Bouwens and Rooman, Physiol Rev 85:1255-1270, 2005).Pdx-1 can be used in the CNIP approach for its post-embryonic action toenhance beta cell formation.

The CNIP approach is designed to induce the post-embryonic developmentof beta cell formation in the adult pancreas without activation of theembryonic pathway of pancreatic development. Others have used theembryonic pathway or stem cells to recreate the embryologic developmentof the pancreas. A disadvantage of inducing the embryonic pathway toaugment beta cell formation is that it induces all of the endocrine celltypes, including glucagon-producing alpha cells that have been shown toplay an important pathogenic role in the glucose intolerance of bothtype 1 diabetes and type 2 diabetes mellitus. The CNIP approach bypassesthe embryonic pathway and stem cell pathway to induce pancreatic betacell formation in the adult pancreas.

Pdx-1 expression and translocation from the cytoplasm to the nucleus areinduced by glucose metabolism signaling to the transcriptional machinery(Mitanchez et al., Endo Rev 18:520-540, 1997). As described, glucosemetabolism is essential for beta cell formation. To create a cell type,the genetic expression profile of the cell has to be changed. Geneinduction by glucose metabolism is an example of an epigenetic phenomenain which the gene expression profile is controlled and regulated by theglucose level in the blood (Doiron et al., J Biol Chem. 271:5321-5324,1996; Mitanchez et al., Endo Rev 18:520-540, 1997). The Pdx-1 geneexpression and translocation from the cytoplasm to the nucleus areinduced by glucose metabolism. However, the effect of glucose metabolismsignaling is to be directed to the transcriptional machinery involvedwith the formation of pancreatic beta cells. The overexpression of Pdx-1will enhance the formation of beta cells by increasing the amount ofPdx-1 that can bind randomly to DNA molecules and find its specific DNAbinding site. Thus, overexpression of Pdx-1 plays a central role inconverging all of the signaling mechanisms in the CNIP cocktail tostimulate beta cell formation. Overexpression of Pdx-1 in the pancreasresults in the convergence of the molecules in the CNIP cocktail toproduce beta cells before activating other effects of glucose metabolismsignaling on the transcriptional machinery that are not related to betacell formation (Doiron et al., J Biol Chem. 271:5321-5324, 1996;Mitanchez et al., Endo Rev 18:520-540, 1997). The Pdx-1 cDNA has beenintroduced into a lentivirus construct using the same method describedabove for lenti-CGK under the control of the CMV promoter. The in vivomethods of injection were used to target the adult mouse pancreas withlenti-CMV-Pdx-1.

Lentivirus CMV-Pdx-1 construct (FIG. 2B): The mouse PDX-1 genes weresubcloned to pEntCMV-WPRE vector and inserts were verified by DNAsequencing. The pENT-PDX-1 were treated with LR Clonase II enzyme(Invitrogen) and ligated to a pLenti vector. The recombination productswere transformed into E. coli cells. After incubation overnight, thepositive clones were selected, and plasmid DNA was purified.

The pEnt-PDX1 and pLenti-PDX1 were transfected into 293 cells. 48 hoursafter transfection, the cells were lysed in SDS-PAGE buffer andsubjected to 4-20% SDS-PAGE gel electrophoresis and Western blotanalyses. The Western blot was carried out using the anti-Myc (for PDX1construct) antibody at a 1:1000 dilution, followed by a HRP conjugatedsecondary antibody. Antibody binding was detected using ECL reagents.

The number of single or two insulin-positive cells in the exocrinetissues were used as an indication of beta cell formation by comparingthe therapeutic group to the control group (FIG. 4). An increase ofsingle or two insulin-positive cells were detected in the exocrinetissues for the therapeutic group.

The increase in single or two insulin positive cells in the therapeuticgroup compared with the control was correlated with the increase in betacell proliferation, quantitated by the marker BrdU (FIG. 5). The BrdUmarker demonstrated proliferation in islets and exocrine tissues.

Co-localization of the proliferation marker BrdU with insulin positivecell demonstrates the formation of new pancreatic beta cells in thegroup injected with the therapeutic composition. Only pancreatic beta(insulin) cell proliferation was observed. No alpha (glucagon) or delta(somatostatin) cell proliferation was detected. By histologicquantification, the therapeutic composition induced pancreatic beta cellformation. Indeed, as explained above, the therapeutic composition wasdesigned to induce the post-embryonic formation of pancreatic beta cellswithout causing the formation of glucagon or somatostatin cells.

Beta cell mass was quantified in the therapeutic and control groups(FIG. 6). Pancreatic beta cell mass was significantly increased in adultmice injected with the therapeutic composition compared with the controladult mice group injected with the control placebo.

Beta cell cluster density in therapeutic group and control group wasquantified. The therapeutic composition caused a significant increase inbeta cell cluster density compared to the control (FIG. 7).

The increase in of pancreatic beta cell proliferation, beta cell mass,and beta cell cluster density was correlated with an increase in insulinproduction after overnight fasting in adult mice injected with thetherapeutic composition compared to the control placebo group (FIG. 8).

B. Methods

In Vivo Method for Targeting Gene Delivery to Adult Pancreas:

To study beta cell formation in the adult animal, the adult pancreas wastargeted in vivo using a viral vector. The methodology has beenvalidated (Doiron et al., Diabetologia 55:719-728, 2012) and employed inthe CNIP approach to generate pancreatic beta cells in vivo.

An advantage of lentiviral vectors is that they do not activatedendritic cells to a significant extent. Furthermore, lentiviral vectorscan (i) infect and integrate into both dividing and nondiving cells,(ii) provide high transduction efficiency and sustain gene expression invivo, (iii) do not induce a significant host immune response, and (iv)can be successfully readministered. Importantly, the method of viralvector injection in vivo into the adult mouse pancreas permits one toevaluate new treatments and/or potential cures for a chronic diseasethat develops in adulthood and avoids the development of compensatorymechanisms that occur when a gene is deleted during embryonicdevelopment. This approach obviates some of the paradoxical findingsthat have been reported with knock out models, i.e. normal/near-normalmuscle insulin sensitivity in mice in whom the insulin receptor isknocked out (See Kitamura et al., Annu. Rev. Physiol., 65:313-32, 2003)and the homozygous null mutant for GLUT4 (GLUT4 −/−)(See Minokoshi etal., Journal of Biol. Chem., 278(36): 33609-12, 2003), which did notmanifest a diabetic phenotype. Therefore, in certain embodiments alentiviral vector is used to target the pancreas directly.

In brief, a lentiviral construct can be introduced into the mousepancreas via the intraductal route, as follows: a 32-gauge catheter(Braintree Scientific, Inc, Braintree, Mass.) is inserted into thecystic duct through a small opening in the gallbladder. The catheter isthen advanced into the common bile duct and secured in place with aslipknot of 0/0 suture around the bile duct and catheter to preventvector reflux into the liver. With a micro clamp placed around thesphincter of Oddi to avoid leakage of the vector into the duodenum, 100μl lentiviral vector expressing green fluorescent protein (GFP) at 10⁸TU/ml is slowly injected into the pancreatic duct through the catheter.Two weeks post-infection, the entire pancreas is removed forhistological examination. After 48 hours, injection of lentivirus codingfor GFP under the control of cytomegalovirus (CMV) promoter specificallytargeted the pancreatic tissues (Doiron et al., 2012). Quantitativemorphometric analysis of pancreatic transduction by the lentivirusvector, based on GFP expression, showed that 60% of the tissue expressedGFP. Expression was detected in the pancreas even after four weeks (FIG.3). The lentivirus vector expressed green fluorescent protein was notfound in any other tissues in the body including heart, lung, liver,brain, leg muscle, and kidney by histology and PCR (data not shown).Pancreatic tissue was stained with H&E to look for evidence ofinflammation (pancreatitis) at day 2 and day 14 post-injection. Noevidence of inflammation was observed. Following the lentiviral vectorinjection containing shRNA Grb10 or shRNA scramble, activity, daily foodintake over the 14 days post-injection; (shRNA scramble mice, 5.4±0.3grams/day [n=5] versus shRNA Grb10 mice, 5.9±0.5 grams/day [n=6]), andweight gain were similar in the shRNA Grb10 and shRNA scramble groups.No diarrhea was observed in either the control or experimental groupsafter the lentivirus injection, and pancreatic (lipase) and hepatic(AST, ALT) enzymes were not elevated (Doiron et al., Diabetologia55:719-728, 2012). These results demonstrate that lentivirus injectiontechnique does not cause adverse gastrointestinal, pancreatic, orhepatic effects.

The inventors also constructed and produced a lentivirus expressingglucokinase (GK), Pdx-1 transcriptional factor, and shRNA targetingPTP1B. Male C57BL/6 mice (Charles River, Wilmington, Mass., USA) 8 weeksof age were used and maintained on an ad libitum diet of water andnormal chow for all experiments. At day 1 post-injection with lentiviralvector, the mice were injected i.p. daily with BrdU (Sigma-Aldrich, StLouis, Mo., USA) in PBS at a dose of 50 μg/g body weight for 12 days toquantitate beta cell proliferation. At 4 weeks post-lentiviralinjection, the entire pancreas was removed for histological examination.

Direct administration to the adult pancreas in vivo can be used toover-produce a protein(s) or to suppress production of a protein(s). Insummary, the inventors have developed a methodological protocol totarget the adult pancreas in vivo. The technique will be employed invalidation studies of the CNIP approach to promote beta cell formation.The lentivirus injection method described above provides proof ofconcept that adult pancreas can be targeted directly. The data obtainedusing the lentivirus approach to target/generate pancreatic beta cellscan be modified using small molecules and non-viral therapeutics.

Animal Studies:

Males C57BL/6 mice (Charles River, Wilmington, Mass., USA) 8 weeks ofage were used and maintained on a diet of water and normal chow adlibitum for all experiments. At 1 day post-injection with lentiviralvector, the mice were injected i.p. daily with BrdU (Sigma-Aldrich, StLouis, Mo., USA) in PBS at a dose of 50 μg/g body weight for 12 days. At4 weeks post-lentiviral injection, the entire pancreas was removed forhistological examination (see below).

Immunofluorescent and Immunohistochemical Analysis:

Adult mouse pancreatic tissues were fixed by immersion in phosphatebuffer 4% paraformaldehyde—1% glutaraldehyde overnight at 4° C. andsubsequently embedded with Tissues-Tek OCT compound for cryostatsectioning. The following primary antibodies were used:anti-somatostatin (G-10), anti-Ki-67 (M-19), anti-glucagon (K79bB10) andanti-insulin A (C-12), antibodies and control rabbit IgG (Santa CruzInc., Santa Cruz, Calif., USA). For proliferation studies, pancreatictissues were stained with either Ki67 (M-19) antibody (Santa Cruz Inc.,Santa Cruz, Calif., USA) or with rat monoclonal BrdU antibody (AbcamInc., Cambridge, Mass., USA). Antigen retrieval was performed for Ki67and BrdU antibodies by boiling sections for 10 min in 10 mM citratebuffer followed by cooling for 30 min to room temperature. Nuclei werecounterstained with DAPI (Vector Laboratories, Inc., Burlingame, Calif.,USA). The fluorescent secondary antibodies used included donkeyanti-goat-fluorescein, goat anti-mouse-fluorescein, goat anti-rabbitTexas red, and donkey anti-goat Texas red (Santa Cruz Inc., Santa Cruz,Calif., USA). The beta cell area represents the surface area of cellsstaining positively for insulin immunostaning divided by the totalpancreatic surface scanned with Olympus FV-1000 laser scanning confocalmicroscope. The insulin positive and total pancreatic areas werequantified with Image J (National Institutes of Health, Bethesda, Md.,USA). Beta cell mass was calculated as beta-cell area multiplied bypancreatic wet weight. At least three mice were analyzed per condition.Pancreatic tissue was stained with H & E to look for evidence ofinflammation (pancreatitis) at day 2 and day 14 post-injection of theLentivirus.

Western Blot:

For western blots, equal amounts of total protein were separated on a 10and 15% SDS/PAGE and transferred onto nitrocellulose membranes.Membranes were then blocked with 5% nonfat milk in 0.1% TBS Tween-20 andprobed with specific antibodies against Pdx-1 (Cell SignalingTechnology, Danvers, Mass., USA), PTP1B (Abcam Inc., Cambridge, Mass.,USA), glucokinase (Santa Cruz Inc, Santa Cruz, Calif., USA), and GAPDH(G9545, Sigma Aldrich, St Louis, Mo., USA). Membranes were thenincubated with HRP-conjugated secondary antibody (NA934) and developedwith a chemiluminescent reagent (Amersham Bioscience, GE Healthcare,Pittsburgh, Pa., USA).

Statistical Analysis:

Results are presented as mean±SEM. Statistical comparisons werepreformed with Student's unpaired t test or one-way ANOVA, whereappropriate. Results were considered to be statistically significantwhen p<0.05.

1. A method for inducing beta cell formation of cells in vitro or in vivo comprising: providing a mammalian cell with a combination of (i) a first agent that increases glucokinase (GK) levels, (ii) a second agent that increases tyrosine receptor kinase activity and/or tyrosine kinase associated receptor activity, and (iii) a third agent that increases Pdx-1 mediated transcription.
 2. The method of claim 1, wherein the mammalian cell is a pancreatic cell, a liver cell, a gut K cell, a neuron, or a stem cell.
 3. The method of claim 1, wherein induction of beta cell formation is in vitro.
 4. The method of claim 3, further comprising isolating a target cell from a subject to be treated.
 5. The method of claim 4, further comprising implanting the treated target cell in the subject to be treated.
 6. The method of claim 3, wherein the mammalian cell is heterologous to the subject to be treated.
 7. A method for inducing beta cell formation from pancreatic cells in vivo comprising: providing to a pancreas in vivo, a combination of (i) a first agent that increases glucokinase (GK) levels, (ii) a second agent that increases tyrosine receptor kinase activity and/or tyrosine kinase associated receptor activity, and (iii) a third agent that increases Pdx-1 mediated transcription in pancreatic cells.
 8. The method of claim 7, wherein the first agent is a nucleic acid encoding glucokinase.
 9. The method of claim 8, wherein the nucleic acid encoding glucokinase is further comprised in a viral vector.
 10. The method of claim 9, wherein the viral vector is a lentivirus vector.
 11. The method of claim 9, further comprising a posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE) 3′ of the coding sequence.
 12. The method of claim 7, wherein the first agent is small molecule activator of glucokinase.
 13. The method of claim 12, wherein the activator of glucokinase is R1440, RO0281675, RO4389620 (Piragliatin), LY2121260, PSN-GK1, or GKA-50.
 14. The method of claim 7, wherein the second agent is an inhibitor of protein tyrosine phosphatase 1B.
 15. The method of claim 14, wherein the protein tyrosine phosphatase 1B (PTP1B) inhibitor is an shRNA inhibitor of protein tyrosine kinase phosphatase 1B or a PTP1B anti-sense DNA.
 16. The method of claim 14, wherein the protein tyrosine phosphatase 1B inhibitor is Wyeth Research Inc., 32D; antisense ISIS-PTP1BRX; Abbott Laboratories, Inc., Isoxazole™; Abbott Laboratories, Inc., antisense oligonucleotides designed to downregulate expression of PTP1B; Merck Frosst Center for Therapeutic Research, selective inhibitors of PTP1B compound 1 and 3; Incyte Corporation, Inc., (S)-isothiazolidinone ((S)-IZD) heterocyclic phosphotyrosine; or Affymax, Inc., triaryl sulfonamide based PTP1B inhibitors.
 17. The method of claim 7, wherein the third agent is a nucleic acid encoding Pdx-1.
 18. The method of claim 7, wherein the first, second, and third agent are provided in a single composition.
 19. The method of claim 7, wherein the first, second, and third agent are provided within a 10 minute administration window.
 20. The method of claim 19, wherein the first, second, and third agent are provided sequentially.
 21. The method of claim 19, wherein the first, second, and third agent are provided simultaneously.
 22. The method of claim 7, wherein the first, second, and third agent are provided by injection of the pancreas through the pancreatic duct.
 23. The method of claim 7, wherein the first and second agent, first and third agent, or the second and third agent are the same.
 24. A method of treating type 1 or type 2 diabetes comprising: providing a therapeutic composition to a pancreas in vivo comprising (i) glucokinase expression cassette configured to express a functional glucokinase protein in a pancreatic cell, (ii) a tyrosine phosphatase 1B inhibitor, and (iii) a Pdx-1 expression cassette configured to express a functional Pdx-1 protein in a pancreatic cell, wherein pancreatic beta cells are induced in the pancreas.
 25. The method of claim 24, wherein diabetes is type 1 diabetes.
 26. The method of claim 24, wherein diabetes is type 2 diabetes. 